A Molecular Dynamics Based Model of the Outward-Facing State and Transport Mechanism of the Human NaCT Homolog Vcindy

VcINDY is a bacterial homolog of the human transporter NaCT, a potential drug target for treatment of obesity and type 2 diabetes. VcINDY is also closely structurally related to the transporter MtrF, which is thought to confer drug resistance to the pathogen that causes gonorrhea. Unlike NaCT, high-resolution crystal structures of VcINDY and MtrF have been solved, but both structures are in inward-facing (IF) states. In this work, we have generated a fully atomistic model of the unknown outward-facing (OF) state of VcINDY embedded stably in a membrane, and we have used this model to structurally characterize a refined transition pathway between the IF and OF states. The conformational differences between the states are experimentally validatable using FRET or DEER measurements, and we have used the OF model to suggest specific cross-link sites to trap VcINDY in an OF state for crystallization. In a new approach, we generated the OF model in the context of a membrane by using nonequilibrium molecular dynamics (MD) simulations to indirectly swap the conformations of the inverted repeats that make up VcINDY. This approach has the immediate benefits of generating a physically stable model and laying the groundwork required to structurally and thermodynamically characterize the full transport cycle of VcINDY. To elucidate the structural transition pathway between the IF and OF states, we used bias-exchange umbrella sampling (BEUS) and the string method with swarms of trajectories (SMwST). Such dynamic structural investigation and further thermodynamic characterization will provide valuable insight into the mechanisms of the biomedically compelling transporters NaCT and MtrF and related proteins, including the other members of the mammalian SLC13 family of transporters and the ∼13,000 members of the AbgT family of transporters.